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Pyrosequencing analysis of KRAS codon 61 mutations in Thai patients with advanced colorectal cancer Cover

Pyrosequencing analysis of KRAS codon 61 mutations in Thai patients with advanced colorectal cancer

Asian Biomedicine
Volume 9 (2015): Issue 1 (February 2015)
By: Chinachote Teerapakpinyo,  Phanni Wanthong,  Mathawee Aumchaaumchaya,  Piyamai Chankate,  Warisa Kaikeaw,  Warunya Tosakorn and  Shanop Shaungshoti  
Open Access
|Jan 2017

Figures & Tables

Figure 1

PCR amplification of exon 3 (codon 61) in KRAS. Codon 61 (CAA, nucleotides 181–183), which resides in exon 3 of KRAS (Transcript ID: ENST00000311936; Ensembl database) is shown in the square box. Mutations in nucleotides 182 and 183 (yellow highlighted nucleotide) reportedly result in amino acid changes including c.182A>T (p.Q61L), c.182A>G (p.Q61R), c.182A>C (p.Q61P), c.183A>C (p.Q61H), and c.183A>T (p.Q61H) in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database, version 63 release. Nucleotides 182 and 183 in codon 61 were sequenced in the reverse direction using KRAS61-PySeq1 and KRAS61-PySeq2 primers. A: KRAS61-Pyseq1 (5′-CTGGTCCCTCATTGCACTGTACTC-3′) and B: KRAS61-Pyseq2 (5′-GTCCCTCATTGCACTGTACTCCTCT-3′) primers complementary to red underlined nucleotides were used for pyrosequencing.
PCR amplification of exon 3 (codon 61) in KRAS. Codon 61 (CAA, nucleotides 181–183), which resides in exon 3 of KRAS (Transcript ID: ENST00000311936; Ensembl database) is shown in the square box. Mutations in nucleotides 182 and 183 (yellow highlighted nucleotide) reportedly result in amino acid changes including c.182A>T (p.Q61L), c.182A>G (p.Q61R), c.182A>C (p.Q61P), c.183A>C (p.Q61H), and c.183A>T (p.Q61H) in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database, version 63 release. Nucleotides 182 and 183 in codon 61 were sequenced in the reverse direction using KRAS61-PySeq1 and KRAS61-PySeq2 primers. A: KRAS61-Pyseq1 (5′-CTGGTCCCTCATTGCACTGTACTC-3′) and B: KRAS61-Pyseq2 (5′-GTCCCTCATTGCACTGTACTCCTCT-3′) primers complementary to red underlined nucleotides were used for pyrosequencing.

Figure 2

Histograms for KRAS codon61 wild-type and mutant discriminations. Histograms (A–G) were generated by PyroMark Q96 software using dispensation order and sequence to analyze as follows; (A–C) 5′-GCTCGATC GA-3′ and 5′-CTC[T/G/A]TGACCTGATGT-3′, (D–G) 5′-TACGTACTGC-3′ and 5′-[T/A/C/G]GACCTGCTG TG-3′, respectively.
Histograms for KRAS codon61 wild-type and mutant discriminations. Histograms (A–G) were generated by PyroMark Q96 software using dispensation order and sequence to analyze as follows; (A–C) 5′-GCTCGATC GA-3′ and 5′-CTC[T/G/A]TGACCTGATGT-3′, (D–G) 5′-TACGTACTGC-3′ and 5′-[T/A/C/G]GACCTGCTG TG-3′, respectively.

Figure 3

Pyrograms showing mutations in KRAS codon 61. (A) Heterozygous c.182A>T (p.Q61L); (B) heterozygous c.183A>T (p.Q61H) and (C) heterozygous c.183A>C (p.Q61H) in samples from Thai patients with advanced colorectal cancer. The red arrows indicate mutant peak and the percentage of mutant allele in the amplified PCR products. wt: wild type; mut: mutant
Pyrograms showing mutations in KRAS codon 61. (A) Heterozygous c.182A>T (p.Q61L); (B) heterozygous c.183A>T (p.Q61H) and (C) heterozygous c.183A>C (p.Q61H) in samples from Thai patients with advanced colorectal cancer. The red arrows indicate mutant peak and the percentage of mutant allele in the amplified PCR products. wt: wild type; mut: mutant
DOI: https://doi.org/10.5372/1905-7415.0901.369 | Journal eISSN: 1875-855X | Journal ISSN: 1905-7415
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Language: English
Page range: 61 - 67
Published on: Jan 31, 2017
Published by: Chulalongkorn University
In partnership with: Paradigm Publishing Services
Publication frequency: 6 issues per year
Keywords:
Colorectal cancer,
KRAS,
mutation,
pyrosequencing,
targeted therapy
Related subjects:
Medicine,
Assistive professions, nursing,
Basic medical science,
Basic medical science, other,
Clinical medicine,
Clinical medicine, other

© 2017 Chinachote Teerapakpinyo, Phanni Wanthong, Mathawee Aumchaaumchaya, Piyamai Chankate, Warisa Kaikeaw, Warunya Tosakorn, Shanop Shaungshoti, published by Chulalongkorn University
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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